Many analysis methods used in biology, chemistry, biotechnology, pharmaceutical and other industrial and research laboratories require accurate measurement and/or calibration of small volumes of liquids. These small volumes can range from nanoliters to microliters. Small volumes of liquid are dispensed from liquid delivery devices, such as pipettes, either sequentially or simultaneously into one or more vessels, such as cuvettes.
In the interest of evaluating a large number of samples of liquid in a desired period of time, multiple charges of liquid samples may be dispensed into a plurality of vessels and analyzed simultaneously or sequentially. Preferably, the plurality of vessels is contained in one or more microtiter plates during testing. A microtiter plate contains a large number of individual wells and, for photometric measurements, a transparent base. Microtiter plates enable the ability to perform more research on a shorter time scale. As a result, they have become the standard analysis platform. The wide use of microtiter plates has resulted in the creation of entire classes of supporting equipment, including various types of analytical instrumentation, such as spectrophotometers capable of measuring individual wells within a plate.
Under the photometric analysis method, the liquid sample under test is contained in the sample vessel and subjected to a light beam of a spectrophotometer. The amount of light from the incident light beam that passes through the vessel and the sample to an opposing detector of the spectrophotometer is dependent upon the characteristics of the liquid sample and the beam path length. It is important that the spectrophotometer provide accurate readings to ensure the accuracy of the liquid sample evaluation.
The Beer-Lambert law defines one useful equation to determine an important characteristic of a sample. Specifically, Beer-Lambert states that the absorbance of light by a liquid sample under test equals the path length traversed by the light beam multiplied by the molar absorptivity of the chromophore in the liquid sample and the concentration of the chromophore in the liquid sample. Knowing the molar absorptivity of the chromophore, and the path length of light, the absorbance measurement provided by the spectrophotometer enables calculation of the particular concentration of the chromophore in the liquid sample. If the path length is not known, or known with sufficient accuracy, the concentration calculated will be approximate, and possibly outside a permitted error range. In addition, if the spectrophotometer is out of specification, the absorbance values obtained from the measurement will be in error and the calculated concentration also in error. It is therefore important to maintain an accurate calibration of the spectrophotometer.
There are several types of spectrophotometers used to measure sample light absorbance. The conventional spectrophotometer transmits a light beam horizontally across the sample vessel. One type of specialized spectrophotometer transmits a light beam vertically through the sample vessel. In horizontal beam spectrophotometers, specific or fixed path lengths can generally be established, as the transmission path length is a function of the fixed cross section of the sample vessel rather than the volume of the sample in the vessel. However, the light transmission and detection method associated with a horizontal beam spectrophotometer is not suitable for use with a microtiter plate arrangement because a filled microtiter plate cannot be properly inserted into the horizontal beam path.
Vertical beam spectrophotometers generally measure solution samples in microtiter plate wells. In one form, the vertical beam spectrophotometer transmits through the sample to a detector on the opposing side of the sample, either from above the sample down through it, or from below up through the sample. The path length for the sample is potentially—and likely—variable from one sample to the next in any given microtiter plate, since the path length is directly dependent upon the volume of sample solution in the well. Other factors that contribute to variations in path length are the ionic strength of the sample solution and the surface characteristics of the plate material, which together define the curvature characteristics of the solution meniscus. All of these factors result in an undefined path length and thus imprecision in vertical beam measurements. Nonetheless, vertical beam measurements are desirable in that multiple samples may be measured more quickly than is possible using a horizontal beam spectrophotometer.
For a horizontal beam spectrophotometer, a common calibration approach used to determine the optical response for a given solution on a given instrument is to either create a series of dilutions of the solution of interest, or measure the solution in different, but known path lengths. In this approach, the ability to use a known path length allows for highly accurate determinations of the optical response of a solution and direct comparison of results from one spectrophotometer to another, or from one solution to another.
One method presently used to calibrate horizontal and vertical beam spectrophotometers involves the measurement of light absorption through a known stable glass filter referred to as a Neutral Density (ND) filter. These filters are pieces of gray-tinted sheet glass that have nearly flat absorbance characteristics over a broad range of wavelengths. ND filters are commonly sent to reference laboratories for certified measurement, which provides results that are traceable to national standards. Once standardized, the ND filters are measured in the horizontal or vertical beam spectrophotometer and the absorbance results are compared to the reference laboratory results as a gauge of accuracy of the instrument.
Although this is a common calibration method, a method that relies on ND filters alone gives little or no information about various optical effects such as: 1) out of band transmission (light passing from the source through the wavelength selection device and the sample to the detector, but outside of the desired wavelength range), 2) wavelength selection accuracy, 3) bandpass of the wavelength selection device, or 4) the shape of the transmission curve of the bandpass selection device. Therefore, reliance exclusively on instrument calibration using ND filters can lead to inaccurate absorbance results, especially when comparison is made between different instruments.
One alternative method for calibrating vertical beam spectrophotometers involves the testing of samples of reference concentrations. Solutions containing different concentrations of the specific dye or chromophore are dispensed into different wells of a microtiter plate. Measurements of optical response are then conducted on the solution-filled wells. This method has several sources of error that limits its usefulness. First, the solution may not obey the Beer-Lambert law exactly, but may slightly deviate from a linear relationship between the concentration of dye and the resulting absorbance of the solution. Second, in order to provide quantitative results, highly accurate control over the amount of liquid dispensed into each well is required. Third, the exact dimensions of the wells in the microtiter plate must be known. Fourth, any meniscus present at the surface of the solution can add to the overall error since it directly affects the path length of light through the solution.
Therefore, what is needed is an apparatus and related method for calibrating vertical beam spectrophotometers. The apparatus should be configured and arranged to be compatible with the arrangement of vertical beam spectrophotometers. The apparatus and method should allow the spectrophotometer operator to account for out of band transmissions, wavelength selection accuracy, the bandpass selection characteristics of the particular spectrophotometer, and the shape of the transmission curve of the bandpass selection. Further, the apparatus and method should resolve deviations in the linear relationship between the concentration of any reference dye in a sample under test and the resulting absorbance of the sample, be independent of the sample volume used to calibrate the spectrophotometer, allow accurate control over the path length, and eliminate meniscus errors in the light beam path.